Broadly neutralizing antibodies (bnAbs) are ideal components for preventing HIV infection. However, no vaccine candidate investigated so far has induced such holy grail components although selected individuals living with HIV have generated bnAbs after a 2 to 4-year delay. We hypothesize that a sequential induction pathway is involved in inducing bnAbs in these infected individuals, and propose new vaccination approaches to mimic the pathway by which HIV induces bnAbs as novel preventive HIV vaccine strategies. We will mimic this process by sequential immunizations with VLPs containing different cEnvs proposed to construct in the project. These cEnvs contain complete different strain-specific epitope profiles but share conserved weakly-immunogenic epitopes for bnAbs. Therefore they will not boost strain-specific immune responses but gradually boost bnAb responses against conserved determinants by sequential immunizations, mimicking the elicitation of bnAbs by the emerging new Envs in evading strain-specific antibody responses in HIV-infected individuals after long term infection, during which strain-specific epitopes undergo continuous antigenic variation. Broadly reactive antibody responses will be evaluated in rabbits. Two specific aims will be pursued: 1) Generation of constructs expressing different cEnvs with conserved regions from the same subtype but variable regions from different subtypes in their gp120s; Production of VLPs of the resulting cEnvs with high incorporation and stabilized Env trimers; 2) Comparison of sequential-immunization regimens with VLPs of various cEnvs for inducing broadly reactive antibody particularly bnAb responses in rabbits by parallel intramuscular and intranasal routes. The goal of this proposal is to develop an effective HIV vaccine strategy to achieve preventive broadly neutralizing antibody responses. This project will provide proof of the concept that sequential-immunization regimen with a panel of VLPs containing cEnv trimers carrying different strain-specific epitopes but sharing conserved epitopes is a potential approach to develop a preventive HIV vaccine.